Vector modifications to eliminate transposase expression following piggyBac-mediated transgenesis

Chakraborty, Samarshi; Ji, HaYeun; Chen, Jack; Gersbach, Charles A.; Leong, Kam W.

doi:10.1038/srep07403

Cited by 8 publications

(8 citation statements)

References 48 publications

(49 reference statements)

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“…The piggyBac transposon system has been successfully used to create random insertion mutant mice 45 69 70 71 72 . However, methods of identifying mice with mutant insertions are not well established.…”

Section: Discussionmentioning

confidence: 99%

PiggyBac transposon-based polyadenylation-signal trap for genome-wide mutagenesis in mice

Liu

Sun

et al. 2016

Sci Rep

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We designed a new type of polyadenylation-signal (PAS) trap vector system in living mice, the piggyBac (PB) (PAS-trapping (EGFP)) gene trapping vector, which takes advantage of the efficient transposition ability of PB and efficient gene trap and insertional mutagenesis of PAS-trapping. The reporter gene of PB(PAS-trapping (EGFP)) is an EGFP gene with its own promoter, but lacking a poly(A) signal. Transgenic mouse lines carrying PB(PAS-trapping (EGFP)) and protamine 1 (Prm1) promoter-driven PB transposase transgenes (Prm1-PBase) were generated by microinjection. Male mice doubly positive for PB(PAS-trapping (EGFP)) and Prm1-PBase were crossed with WT females, generating offspring with various insertion mutations. We found that 44.8% (26/58) of pups were transposon-positive progenies. New transposon integrations comprised 26.9% (7/26) of the transposon-positive progenies. We found that 100% (5/5) of the EGFP fluorescence-positive mice had new trap insertions mediated by a PB transposon in transcriptional units. The direction of the EGFP gene in the vector was consistent with the direction of the endogenous gene reading frame. Furthermore, mice that were EGFP-PCR positive, but EGFP fluorescent negative, did not show successful gene trapping. Thus, the novel PB(PAS-trapping (EGFP)) system is an efficient genome-wide gene-trap mutagenesis in mice.

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Section: Discussionmentioning

confidence: 99%

PiggyBac transposon-based polyadenylation-signal trap for genome-wide mutagenesis in mice

Liu

Sun

et al. 2016

Sci Rep

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“…To create the construct needed for establishing a SSSavi stable expression line (HSB6G), we used Gateway cloning (Invitrogen) to insert dCas9-SSSavi and BirA into a piggyBac plasmid [61]. The dCas9-SSSavi was PCR amplified to include attB sites and then inserted into pDONR221 (Invitrogen) using BP clonase II (Thermo Fisher, #11789020) to create the pENTRY221_dCas9-SSSavi plasmid.…”

Section: Dcas9-sssavi Piggybac Plasmidmentioning

confidence: 99%

A modular dCas9-based recruitment platform for combinatorial epigenome editing

Swain

Pflueger

Freytag

et al. 2022

Preprint

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CRISPR-dCas9 based targeted epigenome editing tools allow precise manipulation and functional investigation of various genome modifications. However, these tools often display substantial context dependency, with highly variable efficacy between target genes and cell types, potentially due to underlying variation in the chromatin modifications present. While simultaneous recruitment of multiple distinct effector chromatin regulators has improved efficacy, these systems typically lack control over which effectors bind and their spatial organisation. To overcome this we have created a new modular combinatorial epigenome editing platform, called SSSavi. This system acts as an interchangeable and reconfigurable docking platform fused to dCas9 to enable simultaneous recruitment of up to four different effectors, allowing precise control and reconfiguration of the effector composition and spatial ordering of their binding. We demonstrate the activity and specificity of the SSSavi system and compare it to existing multi-effector targeting systems, establishing its efficacy. Furthermore, by altering the spatial ordering of effector recruitment, across multiple target genes and cell lines, we demonstrate the importance of effector recruitment order for effective transcriptional regulation. Together, this system offers the capacity to explore effector co-recruitment to specific loci to potentially enhance the manipulation of chromatin contexts previously resistant to targeted epigenomic editing.

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“…The consequence could be saltatory remobilization of the integrated transgene [43]. To limit the effect of sustained transposase expression, a self-inactivated transposase gene has been obtained by including either the promoter [44,45] or the polyadenylation signal [46] between the ITRs ( Table 1). Indeed, in primary human T cells, authors identified an active SB transposase ORF only in one clone out of 94, but a bulk analysis showed up to 0.047 transposase copy integrated per cell [50].…”

Section: Risk Of Transposase Gene Integrationmentioning

confidence: 99%

DNA Elements Tetris: A Strategy for Gene Correction

Bastie¹,

Rouleux‐Bonnin²

2016

Modern Tools for Genetic Engineering

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Transposable elements (TEs) are mobile genetic sequences that are able to move in the genome from one location to another. TEs were first regarded as junk or selfish DNA, as they comprise the largest molecular class within most metazoan genomes having no genomic function. It was necessary to wait until whole genome sequencing to provide new insights about the origin, diversity, and impact of TEs on the genome function. Thus, due to advances in molecular technology, TEs have been shown to create new regulatory sequence networks. Although nowadays most TEs present in the human genome are silenced, particularly DNA transposons, it does not mean that these sequences are dead. In this review, we detail how DNA transposons could be emphasized to create a new tool for gene correction. DNA-based transposon vectors are derived from three models: Sleeping Beauty, piggyBac, and Tol2, which all work via a "cut-and-paste" mechanism where transposase enzyme is alone able to catalyze the transposition process, which means integrating the genes of interest in chromosomal DNA. Limitations and improvements of the systems are discussed, particularly the latest way to target a specific integration site, showing that the DNA transposon-derived system and its engineering, are powerful tools for gene correction.

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Vector modifications to eliminate transposase expression following piggyBac-mediated transgenesis

Cited by 8 publications

References 48 publications

PiggyBac transposon-based polyadenylation-signal trap for genome-wide mutagenesis in mice

PiggyBac transposon-based polyadenylation-signal trap for genome-wide mutagenesis in mice

A modular dCas9-based recruitment platform for combinatorial epigenome editing

DNA Elements Tetris: A Strategy for Gene Correction

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